Elisa Yoko Hirooka

The natural occurrence of fumonisins in Brazilian corn kernels

Since animal intoxication related to corn-based feed is frequently observed in the State of Paraná, Brazil, natural contamination by fumonisins in 48 corn samples (39 from the State of Paraná, and 9 from the Brazilian tropical states, Mato Grosso do Sul and Goias) harvested in 1990-1991 was investigated, along with fungal flora. The total mould count ranged from 6.3 x 10(2) to 5.5 x 10(7) cfu/g, and Fusarium moniliforme and Aspergillus species belonging to section Flavi were detected in 41 and 33 samples, respectively. Regarding the samples from the State of Paraná, F. moniliforme was present in 33 samples at a count of 1.0 x 10(2) to 1.6 x 10(7) cfu/g and Aspergillus spp. in section Flavi in 27 samples at 1.0 x 10(2) to 1.0 x 10(6) cfu/g. HPLC analysis of fumonisins in the corn showed that fumonisins B1 (FB1) and B2 (FB2) were positive for 97.4% and 94.8% of samples respectively. All the corn from North Paraná was positive for fumonisins, with average FB1 levels of 4.79 micrograms/g and average FB2 levels of 3.95 micrograms/g: the Central-West region had average levels of 3.30 and 2.52 micrograms/g, and the Central-East had average of 3.25 and 2.34 micrograms/g, respectively. Except for one negative sample all the corn samples from the Central Region were positive for fumonisins, averaging FB1 levels being 5.45 micrograms/g and FB2 levels being 5.09 micrograms/g. Out of eight samples from the tropical state of Mato Grosso do Sul, F. moniliforme was detected in seven and Aspergillus spp. in section Flavi in five samples with average FB1 levels of 10.59 micrograms/g and average for FB2 levels of 10.31 micrograms/g. The samples from Goias were also contaminated with these two fungi, with the FB1 contamination being 5.83 and the FB2 contamination 3.62 micrograms/g.

Effect of climatic conditions on natural mycoflora and fumonisins in freshly harvested corn of the State of Paraná, Brazil

Natural mycoflora associated with fumonisins were analyzed in 150 samples of freshly harvested corn from Central-Southern, Central-Western and Northern regions of the State of Paraná, Brazil and correlated to climatic conditions. The corn samples were frequently contaminated with Fusarium sp.(98.7 to 100%) and Penicillium sp. (93 to 100%), when compared to Aspergillus sp. (not detected to 27.7%). The highest contamination with potentially mycotoxigenic fungi occurred in corn harvested in the Central-Western region, where total mould and yeast counts ranged from 5.5 × 103 to 5.2 × 106 CFU/g, with 98.7% contaminated byFusarium sp. and 93% by Penicillium sp. In this region F. moniliforme (F. verticillioides) was the predominant Fusariumsp., and was isolated in 85.9% of the samples. Aspergillus sp. was isolated from 27.7% samples. FB1 was detected in 100% of the samples (mean of 2.39 μg/g) and FB2 in 97.7% (mean of 1.09 μg/g). Fumonisins were also detected in all samples from Northern region, with mean of 4.56 μg/g (FB1) and 2.20 μg/g (FB2).Considering 1.0 μg/g as the threshold, 72% of the corn samples from the Central-West and 92% from the North were contaminated with concentrations above this value, in contrast to a 18.5% contamination rate from Central-Southern samples. Between corn planting to harvesting season, the average maximum temperature and relative humidity were 26 °C and 77.1%(Central-Southern), 27 °C and 69% (Northern)and 29.9 °C and 89.1% (Central-Western).Therefore, the higher fumonisins contamination of corn from Northern region when compared to the Central-South were due to the differences in rainfall levels (92.8 mm in Central-Southern, 202 mm in Northern) during the month preceding harvest.

Evaluation of fumonisin-aflatoxin co-occurrence in Brazilian corn hybrids by ELISA

The natural co-occurrence of fumonisins and aflatoxins was investigated in freshly harvested corn kernels (150 samples, 62 hybrids), acquired from the Central-Southern (27 samples, 21 hybrids), Central-Western (86 samples, 51 hybrids) and Northern (37 samples, 18 hybrids) regions of the State of Paraná

Post-harvest storage of corn: effect of beginning moisture content on mycoflora and fumonisin contamination.

The effect of storage on mycoflora profile was monitored bimonthly in 36 corn (Zea mays L.) samples, dividing the same sample into groups dried to 11 and 14% moisture content (1008 analysis). These groups were further subdivided based on the initial total count (moulds and yeasts) up to 104 CFU g-1 (12 samples, range 1.6 × 104 to 9.0 × 104, mean 3.8 × 104 CFU g-1) and up to 105 CFU g-1 (24 samples, range 1.0 × 105 to 5.0 × 105, mean 2.7 × 105 CFU g-1). In the corn group dried to 11%, the fumonisin content was analysed at the initial stage (freshly harvested) and at the end of 12-month storage. Fusarium spp. and Penicillium spp. prevailed at the freshly harvested stage (100%), maintaining this profile throughout 12 months, in corn dried to both 11 and 14%. Cladosporium spp., Aspergillus spp. and Phoma spp. were also detected at lower frequencies during the storage. Fusarium spp. and the total fungal colony count during 12-month storage carried out with samples dried to 11 or 14% moisture content were statistically evaluated using ANOVA for randomized complete block design. The correlation between storage time and Fusarium spp. and total fungal colony count data was analysed by Pearsons correlation test. There was no difference in Fusarium spp. and total counts in the 104 CFU g-1 initial total count group throughout the storage time (p < 0.05). There was a negative correlation between fungal population and storage time (p < 0.05) in the 105 CFU g-1 initial total count group. Fumonisins were detected in all freshly harvested corn, at a mean concentration of 9.9 ± 6.0 µg g-1 (range 0.74-22.6 μg g-1). These values did not change in the 12-month stored corn (mean of 9.9 ± 5.8 μg g-1, range 0.81-23.7 μg g-1). These post harvest data indicated the importance of moisture content at the crop harvesting/predrying stage to control fungal growth and further fumonisin production.

Enterotoxigenicity of Staphylococcus intermedius of canine origin

Seventy-three staphylococcal strains isolated from pyrodermatitis in dogs were classified as Staphylococcus intermedius (52 strains) or Staphylococcus aureus (21 strains) on the basis of acetoin formation, anaerobic mannitol fermentation, aerobic maltose fermentation, pigmentation, coagulation of human plasma, and reaction on crystal violet agar. Enterotoxin was produced by 13 of the 52 S. intermedius strains and 6 of the S. aureus strains. The highest percentage of enterotoxigenic strains produced enterotoxins C (6 strains), D (7 strains), and E (6 strains). Four strains produced the toxic shock syndrome toxin-1. There was little difference in the antibiotic susceptibility between the enterotoxigenic and non-enterotoxigenic strains.

Survey of microcystins in water between 1995 and 1996 in Paraná, Brazil using ELISA.

An enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody was used to determine microcystin (MC) concentrations in water supplies and water plant samples collected between November 1995 and October 1996, from five regions of Paraná, Brazil. In addition, the presence of Microcystis sp. was monitored. Of the 50 samples obtained, 12 were from an urban lake, 8 from human water supplies, 10 from recreational lakes, 13 from farm waters used for animal pasture and 7 from aquaculture facilities. M. aeruginosa was positive in all locations. MCs were positive (>50 pg ml(-1)) in 9 samples (2 samples from human water supplies, 5 from recreational lakes and 2 from animal pasture). Heavy contamination with MCs was observed in water samples collected in May 1996 from 2 recreation (swimming-fishing sites at Itaipu dam, 6380 and 10,000 pg ml(-1)) and human supplies (6627 pg ml(-1)) samples. At these sites, a large bloom of Microcystis sp. was detected. Treatment with 1 ppm Cl- reduced MCs levels, although 267 pg ml(-1) remained in the water plant samples. Our data showed frequent occurrence of Microcystis sp., which may be a hazard to humans and animals in the state of Paraná. More detailed investigations are required to evaluate the risk of natural MC contamination in the water supplied in this region.

Natural occurrence of deoxynivalenol in wheat from Paraná State, Brazil and estimated daily intake by wheat products.

The occurrence of deoxynivalenol (DON) was evaluated in 113 wheat samples from the northern and central/southwestern regions of Paraná State, Brazil during the 2008 and 2009 growing seasons, and this rate of occurrence was used to estimate the DON dietary exposure. The DON determination was carried out by an indirect competitive enzyme-linked immunosorbent assay. DON was detected in 66.4% samples at levels ranging from 206.3 to 4732.3 μg/kg (mean 1894.9 μg/kg). The estimated daily intake (EDI) of DON through bread and pasta was evaluated in the inhabitants of Londrina City in northern Paraná State, Brazil. The average intake of these inhabitants was 0.79 μg/kg body weight (b.w.) for bread and 0.35 μg/kg b.w. for pasta. The total EDI was 1.13 μg/kg, which is above the Provisional Tolerable Daily Maximum Intake (PTDMI) of 1 μg/kg b.w. To our knowledge, this is the first report on natural DON occurrence in wheat and DON dietary exposure estimation from Paraná, Brazil.

Prediction of chicken quality attributes by near infrared spectroscopy

In the present study, near-infrared (NIR) reflectance was tested as a potential technique to predict quality attributes of chicken breast (Pectoralis major). Spectra in the wavelengths between 400 and 2500nm were analysed using principal component analysis (PCA) and quality attributes were predicted using partial least-squares regression (PLSR). PCA performed on NIR dataset revealed the influence of muscle reflectance (L(∗)) influencing the spectra. PCA was not successful to completely discriminate between pale, soft and exudative (PSE) and pale-only muscles. High-quality PLSR were obtained for L(∗) and pH models predicted individually (R(2)CV of 0.91 and 0.81, and SECV of 1.99 and 0.07, respectively). Water-holding capacity was the most challenging attribute to determine (R(2)CV of 0.70 and SECV of 2.40%). Sample mincing and different spectra pre-treatments were not necessary to maximise the predictive performance of models. Results suggest that NIR spectroscopy can become useful tool for quality assessment of chicken meat.

Penicillium expansum versus antagonist yeasts and patulin degradation in vitro

Taking into account the preliminary antagonistic/biodegradation property showed by Pichia membranifaciens and Sporobolomyces roseus, which decreased the initial patulin concentration of 588.4 to 290.0 µg/mL, ability of P. ohmeri 158 in biocontrol against Penicillium expansum and patulin decrease in vitro was performed. The culture supernatant of P. ohmeri 158 was effective against 66.17% micelial growth, indicating antibiosis related with the killer phenomenon. The initial patulin concentration of 223 µg in the presence of P. ohmeri 158 cells was decreased over 83% of the original concentration, when incubated at 25oC/2 days and > 99% after 5 days incubation time, with undetectable patulin level after 15 days. The initial pH 4.0 decreased to pH 3.3 along 15 days experiment, suggesting that patulin decrease was an active process and a consequence of yeast metabolism. The results suggested that P. ohmeri 158 could be a promising alternative for the inhibition of P. expansum growth and patulin degradation.

A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, roasted and instant coffee

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ochratoxin A (OTA) detection in green, roasted and instant coffees was developed using anti-OTA monoclonal antibody. Immunological reagents prepared were OTA-BSA (4.76 mg/mL), anti-OTA.7 MAb (2x103-fold dilution) and HRP-anti IgG (103-fold dilution). The detection limit was 3.73 ng OTA/g and correlation coefficients (r) between this immunoassay and high performance liquid chromatography were 0.98 for green coffee, 0.98 for roasted and 0.86 for instant. OTA levels detected by ic-ELISA were higher than by HPLC, with ELISA/HPLC ratio of 0.66 - 1.46 (green coffee), 0.96 - 1.11 (roasted) and 0.93 - 1.82 (instant). ELISA recoveries for OTA added to coffee (5 - 70 ng/g) were 81.53 % for green coffee, 46.73 % for roasted and 64.35 % for instant, while recoveries by HPLC were 80.54 %, 45.91 % and 55.15 %, respectively. Matrices interferences were minimized by samples dilution before carrying out the ELISA assay. The results indicate that MAb-based ic-ELISA could be a simple, sensitive and specific screening tool for OTA detection, contributing to quality and safety of coffee products.